The present invention relates to methods of identifying combinations of antibodies with a combined improved anti tumor activity. The present invention further relates to pharmaceutical compositions comprising such antibodies which can be used for therapy, such as for cancer therapy.
Receptor tyrosine kinases (RTK) constitute a large family of cell surface proteins that act as scaffolding mediators and molecular switches in many signal transduction pathways, affecting cell growth, proliferation, differentiation, survival and migration. Receptors with tyrosine kinase activity share a similar molecular topology, essentially an extracellular ligand binding domain, a membrane spanning hydrophobic domain, and a cytoplasmic domain that comprises a highly conserved tyrosine kinase catalytic domain. RTKs comprise an array of extracellular domains that bind a variety of growth factors. Characteristically, the extracellular domains are comprised of one or more identifiable structural motifs, including cysteine-rich regions, fibronectin III-like domains, immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like domains, Factor VIII-like domains, glycine-rich regions, leucine-rich regions, acidic regions and discoidin-like domains. Although diverse, RTK activation, initiation of signal transduction, and signal termination follow the same universal model (Yarden, Y., et al., Ann. Rev. Biochem 57:443-478, 1988; Ullrich, A. and Schlessinger, J., Cell 81, 203-212, 1990).
RTKs play a central role in the onset and progression of human disease, particularly cancer, such as breast, colon, lung and prostate cancers. As such, RTKs are preferred targets for the design and configuration of various therapeutic modalities.
Following ligand binding and onset of signaling, ligand-bound receptors are down regulated by removal from the cell surface. This universal mode of down regulation involves ligand-induced internalization by means of endocytosis, primarily via clathrin-coated pits, followed by receptor degradation. RTKs can also be endocytosed from invaginations other than clathrin-coated pits (e.g., caveoli), but the significance of this alternative internalization in RTK downregulation is not yet known. Deregulation of RTK endocytosis is a recurring factor in cancer progenesis (Peschard and Park, Cancer Cell 3: 519-523, 2003; Bache, K. G. et al., EMBO 23: 2707-2712, 2004). Indeed, more than 30 RTKs were found to be associated with cancer, through defective down regulation alone (Blume-Jensen, P. and Hunter, T. Nature 411: 355-365, 2001). Following this and other lines of research, a broad range of RTK inhibitors are being developed for use as anticancer agents or therapeutic agents against other RTK related diseases such as epidermal hyperplasia. RTK inhibitors e.g., epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), Fms-like tyrosine kinase (Flt-3) are constantly being developed [for examples see U.S. Pat. Nos. 6,900,186 (e.g., RTK down regulation for the treatment of skin disorders); 6,399,063 (e.g., down regulation of ErbB-2 with mAb); 5,837,523 (kinase deficient Neu mutants); 5,705,157 (e.g., mAb against EGFR increase receptor down regulation).
While the invention will be described herein in more detail with respect to the ErbB family of RTKs, it is to be understood that the invention is applicable for all RTKs.
The ErbB family of receptor tyrosine kinases, which includes the prototype, epidermal growth factor receptor (EGFR), also termed ErbB-1, and the related proteins ErbB-2, ErbB-3 and ErbB-4 is widely known and researched. The four known members of the ErbB family and their multiple ligand molecules form a layered signaling network, which is implicated in human cancer. Thus, overexpression of ErbB-1/EGF receptor (EGFR) as well as ErbB-2 has been correlated with poor prognosis in various human malignancies. Specifically, deletion mutants of EGFR exist in brain tumors and point mutations have recently been reported in lung cancer. By contrast, ErbB-2/HER2 is rarely mutated in solid tumors. Instead, the ErbB-2 gene is frequently amplified in breast, ovarian, and lung cancer.
Because of their oncogenic potential and accessibility, ErbB proteins have emerged as attractive targets for pharmaceutical interventions. One major strategy involves the use of mAbs. Early studies uncovered the tumor-inhibitory potential of mAbs directed at ErbB-1 and ErbB-2, and later studies indicated that anti-ErbB mAbs are effective when combined with various chemotherapeutic agents. Indeed, the clinical benefit of combining mAbs with certain chemotherapeutic agents was notable, and led to the approval of mAbs to ErbB-2 (Herceptin) and EGFR(C225/Cetuximab) for the treatment of breast and colorectal cancer, respectively. Other antibodies now in clinical trials include MDX-210 (phase II, Medares), tgDCC-Eia (phase II, Targeted Genetics) and 2C4 (phase I, Genentech) (Zhang H. et al., Cancer Biol. Therap., 2: S122-S126, 2003). Unfortunately, the therapeutic efficacy of these and other RTK inhibitors is limited and varies dramatically between patients. There is thus a need to elucidate the mechanism underlying antibody mediated therapy.
Two types of mechanisms have been implicated in ErbB-directed immunotherapy. The first involves mAb-mediated recruitment of natural killer cells through the Fc-γ activation receptors of these immune effector cells to the tumor site. The second type of mechanism relates to intrinsic mAb activities, which include blockade of ligand binding or receptor pharmaceutical composition comprising as an active ingredient at least two anti RTK antibodies capable of inducing synergistic endocytosis of the RTK in a cell expressing the RTK.
According to still another aspect of the present invention there is provided a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of at least two anti RTK antibodies capable of inducing synergistic endocytosis of the RTK in a cell expressing the RTK, thereby treating cancer in the subject.
According to further features in preferred embodiments of the invention described below, the at least two antibodies are directed to distinct extracellular epitopes of the RTK.
According to still further features in the described preferred embodiments each of the at least two antibodies bind the RTK with an affinity of at least 200 nM.
According to still further features in the described preferred embodiments the at least two antibodies bind the RTK with a similar affinity.
According to still further features in the described preferred embodiments the RTK is an ErbB protein.
According to still further features in the described preferred embodiments the ErbB protein is selected from the group consisting of ErbB-1, ErbB-2, ErbB-3 and ErbB-4.
According to still further features in the described preferred embodiments the RTK is selected from the group consisting of c-met, PDGFR, ErbB, VEGFR, EphR, FGFR, INSR and AXL.
According to still further features in the described preferred embodiments the at least two antibodies comprise a recombinant antibody.
According to still further features in the described preferred embodiments the recombinant antibody is a bispecific antibody or a single chain antibody.
According to still further features in the described preferred embodiments the at least two antibodies comprise a monoclonal antibody.
According to still further features in the described preferred embodiments the at least two antibodies comprise a polyclonal antibody.
According to still further features in the described preferred embodiments the at least two antibodies comprise a humanized antibody.
According to still further features in the described preferred embodiments the at least two antibodies comprise an antibody fragment.
According to still further features in the described preferred embodiments the at least two antibodies comprise at least a bivalent antibody.
According to still further features in the described preferred embodiments the endocytosis is c-Cbl independent.
According to still further features in the described preferred embodiments the endocytosis is ubiquitylation independent.
According to still further features in the described preferred embodiments the method further comprising subjecting the subject to a therapy selected from the group consisting of a radiotherapy and a chemotherapy.
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of identifying combinations of antibodies with a combined improved anti tumor activity.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.